Multiple beads per droplet resolution

ABSTRACT

Methods of generating a nucleic acid signature for identifying particles associated in a partition are provided. In one aspect, the method comprises: partitioning a sample into a plurality of partitions comprising a particle comprising a solid support surface, the solid support surface having a plurality of oligonucleotide primers conjugated thereon, wherein the oligonucleotide primers comprise a barcode sequence, and wherein the partitions have 0, 1, or more than 1 particles per partition; providing in a partition a substrate comprising a barcode sequence or repeating clonal barcode sequences; and in the partition, associating a first particle conjugated to oligonucleotide primers comprising a first barcode sequence and a second particle conjugated to oligonucleotide primers comprising a second barcode sequence to a barcode sequence from the substrate, thereby generating a nucleic acid signature for the particles in the partition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/911,961 filed Jun. 25, 2020, which is a continuation of U.S.application Ser. No. 15/400,756, filed Jan. 6, 2017; now U.S. Pat. No.10,730,030 issued Aug. 4, 2020, which claims priority to U.S.Provisional Application No. 62/276,592, filed Jan. 8, 2016, the contentsof each of which are incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

Beads conjugated to oligonucleotides are used in microfluidic detectionapplications such as high-throughput sequencing. In order to uniquelyidentify each partition, the beads can be labeled with unique barcodesequences. However, in order to ensure that partitions have only onebead and thus are uniquely labeled by the barcode, bead concentrationsare typically adjusted so that only about 1 out of 10 partitions areoccupied by a bead. This results in about 90% dead volume in thepartitions, and increases the amount of sample and reagents that isneeded for detection of samples.

BRIEF SUMMARY OF THE INVENTION

In one aspect, methods of generating a nucleic acid signature foridentifying particles associated in a partition are provided. In someembodiments, the method comprises:

-   -   (a) partitioning a sample into a plurality of partitions        comprising a particle comprising a solid support surface, the        solid support surface having a plurality of oligonucleotide        primers conjugated thereon, wherein the oligonucleotide primers        comprise a barcode sequence and wherein at least a majority of        the plurality of oligonucleotide primers conjugated to a solid        support surface comprise the same barcode sequence, and wherein        the partitions have 0, 1, or more than 1 particles per        partition;    -   (b) providing in a partition a substrate comprising a barcode        sequence or repeating clonal barcode sequences; and    -   (c) in the partition, associating a first particle conjugated to        oligonucleotide primers comprising a first barcode sequence and        a second particle conjugated to oligonucleotide primers        comprising a second barcode sequence to a barcode sequence from        the substrate; thereby generating a nucleic acid signature for        the particles in the partition.

In some embodiments, the providing step comprises releasing the barcodesequence or the repeating clonal barcode sequences from the substrate.In some embodiments, the substrate comprising repeating clonal barcodesequences comprises tandem repeating clonal barcode sequences that areseparated by a cleavable linker. In some embodiments, the releasing stepcomprises cleaving the substrate at the cleavable linker. In someembodiments, the cleavable linker is a restriction enzyme recognitionsite, a uridine incorporated site, or a photocleavable nucleotide.

In some embodiments, the substrate comprising the barcode sequence orrepeating clonal barcode sequences is a droplet encapsulating therepeating clonal barcode sequences, and the providing step comprisesreleasing the barcode sequence or the repeating clonal barcode sequencesfrom the substrate. In some embodiments, the releasing step comprisesbreaking the droplet.

In some embodiments, the substrate barcode sequence or repeating clonalbarcode sequences comprise DNA, RNA, or a DNA/RNA hybrid. In someembodiments, the substrate barcode sequence or repeating clonal barcodesequences are single-stranded (e.g., single-stranded DNA or RNA). Insome embodiments, the substrate barcode sequence or repeating clonalbarcode sequences are double-stranded (e.g., double-stranded DNA, RNA,or DNA/RNA hybrid). In some embodiments, the substrate barcode sequenceis a contiguity preserved tagmented polynucleotide sequence.

In some embodiments, the substrate comprises repeating clonal barcodesequences. In some embodiments, the substrate comprising the repeatingclonal barcode sequences is a hairpin molecule. In some embodiments, thesubstrate comprising the repeating clonal barcode sequences is a linearpolynucleotide substrate. In some embodiments, the substrate comprisingthe repeating clonal barcode sequences is a circular polynucleotidesubstrate. In some embodiments, the circular polynucleotide substrate isa plasmid, a DNA nanoball, or a multiple displacement amplified branchedsubstrate.

In some embodiments, the barcode sequence or repeating barcode sequenceof the substrate has a length of at least 6 nucleotides. In someembodiments, the repeating clonal barcode sequences comprise at least 2,at least 5, at least 10, at least 50, or at least 100 repeats of theclonal barcode sequence.

In some embodiments, the substrate comprises a single barcode sequence.In some embodiments, the associating step comprises (1) annealing anoligonucleotide primer of the first particle to the substrate barcodesequence and extending the annealed product with a polymerase, and (2)annealing an oligonucleotide primer of the second particle to thesubstrate barcode sequence and extending the annealed product with thepolymerase.

In some embodiments, the providing step comprises providing in apartition a plurality of substrates comprising a barcode sequence orrepeating clonal barcode sequences, wherein the plurality of substratescomprise distinguishable barcode sequences. In some embodiments, theproviding step comprises providing at least 10, at least 20, at least30, at least 40, at least 50, at least 100, or at least 200 substratescomprising a barcode sequence or repeating barcode sequence.

In some embodiments, the partitions have an average of at least about0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3 or more particles perpartition. In some embodiments, the partitions are droplets. In someembodiments, the partitions have an average of about 1 particle perpartition. In some embodiments, the partitions have an average of about2 particles per partition. In some embodiments, the partitions have anaverage of about 3 particles per partition.

In some embodiments, the associating step comprises associating (1) thefirst particle conjugated to oligonucleotide primers comprising thefirst barcode sequence with a first substrate barcode sequence, and (2)the second particle conjugated to oligonucleotide primers comprising thesecond barcode sequence with a second substrate barcode sequence. Insome embodiments, the associating step the associating step comprisesannealing the first substrate barcode sequence to an oligonucleotideprimer of the first particle and the second substrate barcode sequenceto an oligonucleotide primer of the second particle and extending theannealed products with a polymerase.

In some embodiments, the associating step comprises at least 1 cycle ofannealing and extension. In some embodiments, the associating stepcomprises at least 5, at least 10, at least 15, at least 20, at least25, at least 30, at least 35, or at least 40 cycles of annealing andextension.

In some embodiments, the first substrate barcode sequence and the secondsubstrate barcode sequence are from different substrates. In someembodiment, the first substrate barcode sequence and the secondsubstrate barcode sequence are from the same substrate.

In some embodiments, the associating step comprises ligating a substratebarcode sequence to both an oligonucleotide primer of the first particleand an oligonucleotide primer of the second particle.

In some embodiments, the sample comprises a target nucleic acid. In someembodiments, the sample comprising a target nucleic acid comprises acell containing the target nucleic acid.

In another aspect, a plurality of partitions generated according to themethods described herein are provided.

In another aspect, partition libraries are provided. In someembodiments, the partition library comprises two or more partitions,wherein at least some partitions comprise at least a first particleconjugated to oligonucleotide primers comprising a first barcodesequence and a second particle conjugated to oligonucleotide primerscomprising a second barcode sequence, and further comprise a substratecomprising a barcode sequence or repeating clonal barcode sequences.

In some embodiments, the substrate comprises a single barcode sequence.In some embodiments, the substrate comprises repeating clonal barcodesequences. In some embodiments, the substrate comprising the repeatingclonal barcode sequences comprises tandem repeating clonal barcodesequences that are separated by a cleavable linker. In some embodiments,the substrate comprising the tandem repeating clonal barcode sequencesis a hairpin molecule. In some embodiments, the substrate comprising thetandem repeating clonal barcode sequences is a linear polynucleotidesubstrate. In some embodiments, the substrate comprising the tandemrepeating clonal barcode sequences is a circular polynucleotidesubstrate. In some embodiments, the substrate comprising the repeatingclonal barcode sequences is a droplet encapsulating the repeating clonalbarcode sequences.

In some embodiments, the first substrate barcode sequence and the secondsubstrate barcode sequence are distinguishable sequences. In someembodiments, the first substrate barcode sequence and the secondsubstrate barcode sequence are identical sequences.

In some embodiments, the partition library comprises a plurality ofsubstrates comprising a barcode sequence or repeating clonal barcodesequences, wherein the plurality of substrates comprise distinguishablebarcode sequences.

In some embodiments, the partition library comprises two or morepartitions, wherein at least some partitions comprise at least a firstparticle conjugated to oligonucleotide primers comprising a firstbarcode sequence and a second particle conjugated to oligonucleotideprimers comprising a second barcode sequence, and further comprise atleast one substrate barcode sequence associated with the first particleand the second particle. In some embodiments, at least some partitionscomprise a first substrate barcode sequence associated with the firstparticle conjugated to oligonucleotide primers comprising the firstbarcode sequence and a second substrate barcode sequence associated withthe second particle conjugated to oligonucleotide primers comprising thesecond barcode sequence. In some embodiments, the first substratebarcode sequence and the second substrate barcode sequence aredistinguishable sequences. In some embodiments, the first substratebarcode sequence and the second substrate barcode sequence are identicalsequences. In some embodiments, at least some partitions comprise asubstrate barcode sequence ligated to both an oligonucleotide primer ofthe first particle and to an oligonucleotide primer of the secondparticle.

In some embodiments, a partition library comprises at least 500, atleast 1000, at least 10,000, at least 50,000, or at least 100,000partitions. In some embodiments, the partitions comprise droplets. Insome embodiments, the partitions have an average of at least about 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3 or more particles per partition.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, analytical chemistry,and nucleic acid chemistry and hybridization described below are thosewell-known and commonly employed in the art. Standard techniques areused for nucleic acid and peptide synthesis. The techniques andprocedures are generally performed according to conventional methods inthe art and various general references (see generally, Sambrook et al.MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., which is incorporated hereinby reference), which are provided throughout this document.

The term “amplification reaction” refers to any in vitro method formultiplying the copies of a target sequence of nucleic acid in a linearor exponential manner. Such methods include, but are not limited to,polymerase chain reaction (PCR); DNA ligase chain reaction (LCR); QBetaRNA replicase and RNA transcription-based amplification reactions (e.g.,amplification that involves T7, T3, or SP6 primed RNA polymerization),such as the transcription amplification system (TAS), nucleic acidsequence based amplification (NASBA), and self-sustained sequencereplication (3SR); single-primer isothermal amplification (SPIA), loopmediated isothermal amplification (LAMP), strand displacementamplification (SDA); multiple displacement amplification (MDA); rollingcircle amplification (RCA); as well as others known to those of skill inthe art. See, e.g., Fakruddin et al., J. Pharm Bioallied Sci. 20135(4):245-252.

“Amplifying” refers to a step of submitting a solution to conditionssufficient to allow for amplification of a polynucleotide if all of thecomponents of the reaction are intact. Components of an amplificationreaction include, e.g., primers, a polynucleotide template, polymerase,nucleotides, and the like. The term “amplifying” typically refers to an“exponential” increase in target nucleic acid. However, “amplifying” asused herein can also refer to linear increases in the numbers of aselect target sequence of nucleic acid, such as is obtained with cyclesequencing or linear amplification.

“Polymerase chain reaction” or “PCR” refers to a method whereby aspecific segment or subsequence of a target double-stranded DNA, isamplified in a geometric progression. PCR is well known to those ofskill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; andPCR Protocols: A Guide to Methods and Applications, Innis et al., eds,1990. Exemplary PCR reaction conditions typically comprise either two orthree step cycles. Two step cycles have a denaturation step followed bya hybridization/elongation step. Three step cycles comprise adenaturation step followed by a hybridization step followed by aseparate elongation step.

A “primer” refers to a polynucleotide sequence that hybridizes to asequence on a target nucleic acid and serves as a point of initiation ofnucleic acid synthesis. Primers can be of a variety of lengths. In someembodiments, a primer is less than 50 nucleotides in length, e.g., fromabout 10 to about 40, from about 15 to about 40, or from about 15 toabout 30 nucleotides in length. The length and sequences of primers foruse in an amplification reaction (e.g., PCR) can be designed based onprinciples known to those of skill in the art; see, e.g., PCR Protocols:A Guide to Methods and Applications, Innis et al., eds, 1990. In someembodiments, a primer comprises one or more modified or non-naturalnucleotide bases. In some embodiments, a primer comprises a label (e.g.,a detectable label).

A nucleic acid, or portion thereof, “hybridizes” to another nucleic acidunder conditions such that non-specific hybridization is minimal at adefined temperature in a physiological buffer. In some cases, a nucleicacid, or portion thereof, hybridizes to a conserved sequence sharedamong a group of target nucleic acids. In some cases, a primer, orportion thereof, can hybridize to a primer binding site if there are atleast about 6, 8, 10, 12, 14, 16, or 18 contiguous complementarynucleotides, including “universal” nucleotides that are complementary tomore than one nucleotide partner. Alternatively, a primer, or portionthereof, can hybridize to a primer binding site if there are fewer than1 or 2 complementarity mismatches over at least about 12, 14, 16, or 18contiguous complementary nucleotides. In some embodiments, the definedtemperature at which specific hybridization occurs is room temperature.In some embodiments, the defined temperature at which specifichybridization occurs is higher than room temperature. In someembodiments, the defined temperature at which specific hybridizationoccurs is at least about 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80°C.

As used herein, “nucleic acid” refers to DNA, RNA, single-stranded,double-stranded, or more highly aggregated hybridization motifs, and anychemical modifications thereof. Modifications include, but are notlimited to, those providing chemical groups that incorporate additionalcharge, polarizability, hydrogen bonding, electrostatic interaction,points of attachment and functionality to the nucleic acid ligand basesor to the nucleic acid ligand as a whole. Such modifications include,but are not limited to, peptide nucleic acids (PNAs), phosphodiestergroup modifications (e.g., phosphorothioates, methylphosphonates),2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, methylations, unusual base-pairing combinationssuch as the isobases, isocytidine and isoguanidine and the like. Nucleicacids can also include non-natural bases, such as, for example,nitroindole. Modifications can also include 3′ and 5′ modificationsincluding but not limited to capping with a fluorophore (e.g., quantumdot) or another moiety.

As used herein, the term “partitioning” or “partitioned” refers toseparating a sample into a plurality of portions, or “partitions.”Partitions can be solid or fluid. In some embodiments, a partition is asolid partition, e.g., a microchannel. In some embodiments, a partitionis a fluid partition, e.g., a droplet. In some embodiments, a fluidpartition (e.g., a droplet) is a mixture of immiscible fluids (e.g.,water and oil). In some embodiments, a fluid partition (e.g., a droplet)is an aqueous droplet that is surrounded by an immiscible carrier fluid(e.g., oil).

As used herein, a “barcode” is a short nucleotide sequence (e.g., atleast about 4, 6, 8, 10, 12, or 14 nucleotides long) that identifies amolecule to which it is conjugated. In some embodiments, a barcode isused to identify molecules in a partition. Such a partition-specificbarcode should be unique for that partition as compared to barcodespresent in other partitions. For example, partitions containing targetRNA from single cells can subject to reverse transcription conditionsusing primers that contain a different partition-specific barcodesequence in each partition, thus incorporating a copy of a unique“cellular barcode” into the reverse transcribed nucleic acids of eachpartition. Thus, nucleic acid from each cell can be distinguished fromnucleic acid of other cells due to the unique “cellular barcode.” Insome embodiments, a barcode is present on oligonucleotides conjugated toa particle, wherein the “particle barcode” is shared by (e.g., identicalor substantially identical amongst) all, or substantially all, of theoligonucleotides conjugated to that particle.

In some embodiments, a barcode is provided in a substrate. In someembodiments, a substrate comprises a single barcode sequence. In someembodiments, a substrate comprises repeating clonal barcode sequences.As used herein, a “substrate comprising repeating clonal barcodesequences” refers to a composition that contains a plurality ofidentical barcode sequences that are physically connected to each other(e.g., in a hairpin molecule, a linear nucleic acid polymer, or acircular nucleic acid polymer as tandem repeating barcode sequencesseparated by a cleavable linker between each repeating sequence) or thatare sequestered from other components when delivered to a partition(e.g., encapsulated within a droplet that is delivered to a partition).In some embodiments, individual barcode sequences are released from thesubstrate or released from a repeating clonal barcode sequence (e.g.,released from the hairpin, linear nucleic acid polymer, or circularnucleic acid polymer by cleaving the polymer at the cleavable linkers,or released from the droplet by breaking the droplet) in a partition,and are associated with a particle in the partition, for example, byannealing the clonal barcode sequence to an oligonucleotide on thepartition or by ligating the clonal barcode sequence to anoligonucleotide on the partition.

As used herein, the term “nucleic acid signature” refers to a uniquecombination of multiple distinguishable barcodes that is associated witha partition or a combination of particles within a partition. In someembodiments, a nucleic acid signature is used to identify a partitionassociated with a target nucleic acid (e.g., in a sequencing read, toidentify the partition from which a sequenced target nucleic acidoriginated).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C. Schematic depicting an exemplary process of generating anucleic acid signature for particles in partitions. (A) A firstpartition (“Drop 1”) comprises two particles (“bead1” and “bead2”) witholigonucleotides conjugated to the particles, each particle having adistinguishable barcode on its oligonucleotides (“bc1” and “bc2,”respectively). The first partition further comprises two substratescomprising repeating clonal barcode sequences in the form of hairpinoligos, each substrate having a distinguishable barcode (barcode “5” inred, and barcode “6” in purple). A second partition (“Drop 2”) comprisestwo particles (“bead3” and “bead4”) with oligonucleotides conjugated tothe particles, each particle having a distinguishable barcode (“bc3” and“bc4,” respectively). The second partition further comprises twosubstrates comprising repeating clonal barcode sequences in the form ofhairpin oligos, each substrate having a distinguishable barcode (barcode“7” in blue, and barcode “8” in green). Each partition further comprisesone target molecule per partition. The cleavable linkers in the hairpinoligos are cleaved, releasing the individual clonal barcode sequences inthe partition. (B) The partitions are subjected to denaturationconditions that denature the double-stranded clonal barcode sequencesinto single-stranded clonal barcode sequences. After an annealing andextension reaction, different combinations of clonal barcode sequencesassociated with particle-conjugated oligonucleotides are possible ineach partition. (C) The combinations of the clonal barcode associatedwith particle-conjugated oligonucleotides yield a nucleic acid signaturefor each partition. For Drop 1, bead barcodes 1 and 2 will only appearwith clonal barcode sequences 5 and 6. For Drop 2, bead barcodes 3 and 4will only appear with clonal barcode sequences 7 and 8. In sequencingreads, the presence of a nucleic acid signature associated with aparticular partition will indicate the partition from which a targetmolecule originated.

FIG. 2 . Substrates comprising repeating clonal barcode sequences can becircular substrates, including single stranded nucleic acid polymers(RNA, DNA, or a mixture thereof) and double stranded nucleic acidpolymers (RNA, DNA, or a mixture thereof). The oligonucleotidesconjugated to the particles and the substrate barcode sequences compriseuniversal tag sequences (tag B, tag C) for hybridizing the substratebarcode sequences to the particle-conjugated oligonucleotides.

FIG. 3 . Substrates comprising repeating clonal barcode sequences can belinear substrates, including single stranded nucleic acid polymers (RNA,DNA, or a mixture thereof) and double stranded nucleic acid polymers(RNA, DNA, or a mixture thereof).

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Described herein are methods, compositions, and kits for generating anucleic acid signature for identifying particles that are associatedwith each other in a partition. In a partition comprising two or moreparticles conjugated to barcode sequence-containing oligonucleotideprimers (e.g., partitions comprising two particles or partitionscomprising three particles), one or more additional barcode sequencesare introduced into the partition that are distinct from the barcodes ofthe oligonucleotide primers conjugated to the particles. In someembodiments, the additional barcode sequences are introduced into apartition in a substrate. The substrate barcode sequences are associatedwith the oligonucleotide primers (e.g., in annealing and extensionreactions, ligation, or amplification reactions) to generate acombination of barcode sequences that is unique to the particles in thatpartition. This unique combination of barcodes for the particles in apartition is a “nucleic acid signature” that identifies the particles asoriginating from a specific partition.

Nucleic acid signatures for partitions can be used, e.g., fordeconvoluting data from a pool of partitions. For example, in someembodiments, the contents of multiple partitions are pooled fordetection (e.g., by sequencing), and the nucleic acid signature can beused to deconvolute the sequencing data in order to identify thespecific partition from which a target nucleic acid originated.

The methods, compositions, kits, and partition libraries describedherein can be used, e.g., for increasing the rate of occupancy forpartitions loaded with particles. As described herein, the use of two orthree particles per partition on average increases particle occupancyfrom about 10% to about 85% or 95%, thereby drastically minimizing thedead volume of partitions and improving the efficiency ofpartition-based reactions.

II. Methods of Generating a Nucleic Acid Signatures for Particles in aPartition

In one aspect, methods of generating a nucleic acid signature foridentifying particles associated in a partition are provided. In someembodiments, the method comprises:

-   -   (a) partitioning a sample into a plurality of partitions        comprising a particle comprising a solid support surface, the        solid support surface having a plurality of oligonucleotide        primers conjugated thereon, wherein the oligonucleotide primers        comprise a barcode sequence and wherein at least a majority of        the plurality of oligonucleotide primers conjugated to a solid        support surface comprise the same barcode sequence, and wherein        the partitions have 0, 1, or more than 1 particles per        partition;    -   (b) providing in a partition a substrate comprising a barcode        sequence or repeating clonal barcode sequences; and    -   (c) in the partition, associating a first particle conjugated to        oligonucleotide primers comprising a first barcode sequence and        a second particle conjugated to oligonucleotide primers        comprising a second barcode sequence to a clonal barcode        sequence;    -   thereby generating a nucleic acid signature for the particles in        the partition.

In some embodiments, the sample to be partitioned comprises one or moretarget nucleic acids. In some embodiments, the sample to be partitionedcomprises one or more target nucleic acids and further comprisesparticles conjugated to the oligonucleotide primers and/or substratescomprising repeating clonal barcode sequences.

Samples

In some embodiments, the method comprises partitioning a samplecomprising one or more target nucleic acids into a plurality ofpartitions. In some embodiments, the sample comprising target nucleicacids comprises DNA, RNA, or a combination or hybrid thereof. In someembodiments, the sample comprising target nucleic acids comprisesgenomic DNA or DNA from a subset of a genome (e.g., selected genes thatmay harbor mutations for a particular population, such as individualswho are predisposed for a particular type of cancer). In someembodiments, the sample comprising target nucleic acids comprises cDNA.In some embodiments, the sample comprising target nucleic acidscomprises exome DNA (i.e., a subset of whole genomic DNA enriched fortranscribed sequences which contains the set of exons in a genome) ortranscriptome DNA (i.e., the set of all mRNA or “transcripts” producedin a cell or population of cells). In some embodiments, the samplecomprising target nucleic acids comprises long fragment DNA (e.g., DNAhaving a length of at least about 300, 400, 500, 600, 700, 800, 1000, ormore bases, or base pairs for double-stranded DNA). In some embodiments,the sample comprising target nucleic acids comprises RNA, e.g., mRNA orlncRNA. In some embodiments, the target nucleic acids are doublestranded. In some embodiments, the target nucleic acids are singlestranded. In some embodiments, the sample comprises target nucleic acidsisolated from tissue, cells, or a single-cell sample.

In some embodiments, the sample comprising target nucleic acids is abiological sample. Biological samples can be obtained from anybiological organism, e.g., an animal, plant, fungus, pathogen (e.g.,bacteria or virus), or any other organism. In some embodiments, thebiological sample is from an animal, e.g., a mammal (e.g., a human or anon-human primate, a cow, horse, pig, sheep, cat, dog, mouse, or rat), abird (e.g., chicken), or a fish. A biological sample can be any tissueor bodily fluid obtained from the biological organism, e.g., blood, ablood fraction, or a blood product (e.g., serum, plasma, platelets, redblood cells, and the like), sputum or saliva, tissue (e.g., kidney,lung, liver, heart, brain, nervous tissue, thyroid, eye, skeletalmuscle, cartilage, or bone tissue); cultured cells, e.g., primarycultures, explants, and transformed cells, stem cells, stool, urine,etc. In some embodiments, the sample is a sample comprising cells. Insome embodiments, the sample is a single-cell sample.

In some embodiments, the methods described herein are used for singlecell analysis. Accordingly, in some embodiments, target nucleic acidsfrom a single cell are partitioned into a plurality of partitions. Insome embodiments, target nucleic acids from a biological samplecontaining a plurality of cells are extracted and partitioned such thatindividual partitions contain nucleic acid from less than one, one, or aplurality of cells.

Particles Conjugated to Oligonucleotide Primers

In some embodiments, particles that are conjugated to barcode-labeledoligonucleotide primers are used in the methods, partition libraries,and kits described herein. In some embodiments, the particle comprises asolid support surface having a plurality of oligonucleotide primersconjugated thereon. In some embodiments, the particle comprises at leastabout 10, 50, 100, 500, 1000, 5000, 10,000, 50,000, 100,000, 500,000,1,000,000, 5,000,000, 10,000,000 or more oligonucleotide primersconjugated thereto. In some embodiments, the oligonucleotide primers aredouble stranded. In some embodiments, the oligonucleotide primers aresingle stranded.

In some embodiments, the oligonucleotide primers comprise a barcodesequence, wherein at least a majority, substantially all, or all of theplurality of oligonucleotide primers conjugated to a solid supportsurface comprise the same barcode sequence. In some embodiments, thebarcode is a sequence of about 6 to about 20 nucleotides, e.g., about6-16, about 6-14, about 8-20, about 8-18, about 10-20, about 10-18, orabout 12-20 nucleotides. In some embodiments, the barcode is a sequenceof at least about 10 nucleotides. In some embodiments, theoligonucleotide primers conjugated to a particular particle comprise abarcode sequence that is the same or substantially the same among theplurality of oligonucleotides on a particle, but unique or substantiallyunique as compared to the plurality of oligonucleotides on otherparticles.

In some embodiments, the oligonucleotide primers comprise a “tag”portion. In some embodiments, the tag portion provides a functionalityto be used for downstream steps. For example, in some embodiments, thetag portion comprises a universal sequence that is common to all orsubstantially all oligonucleotide primers on all particles. In someembodiments, the tag portion comprises a primer for use in a downstreamamplification step. In some embodiments, the tag portion comprises asequence or a partial sequence of a sequencing adapter, e.g., a RD1sequence from the P5 adapter (Illumina), or a sequence complementary tothe adapter sequence or portion of the adapter sequence (e.g., RD1sequence). In some embodiments, the tag portion is at the 5′ end of theoligonucleotide primer.

In some embodiments, the oligonucleotide primers comprise a randomsequence portion. In some embodiments, the random sequence portion of anoligonucleotide primer is used for hybridizing to a target nucleic acidor a clonal barcode sequence and/or is used as a primer in a downstreamprimer extension step. In some embodiments, the random sequence portionis a sequence of at least about 5, 10, 15 or more nucleotides, e.g.,about 6-8, about 6-10, or about 5-15 nucleotides.

In some embodiments, the oligonucleotide primers comprise a poly-thymineregion. In some embodiments, the poly-thymine region comprises fromabout 15 to about 20, or from about 15 to about 35 thymine nucleotides.In some embodiments, the poly-thymine region is at the 3′ end of theoligonucleotide primer.

In some embodiments, the plurality of oligonucleotide primers areconjugated to the solid support surface of the particle at the 5′ end ofthe oligonucleotide primer. In some embodiments, the oligonucleotideprimers comprise a barcode sequence portion in the middle portion of theoligonucleotide primer and a 3′ end that is available for ligationand/or extension by a polymerase.

Solid supports suitable for attaching oligonucleotides thereto includecontrolled pore glass (CPG) (available from Glen Research, Sterling,Va.), oxalyl-controlled pore glass (See, e.g., Alul, et al., NucleicAcids Research 1991, 19, 1527), TentaGel Support—anaminopolyethyleneglycol derivatized support (See, e.g., Wright, et al.,Tetrahedron Letters 1993, 34, 3373), polystyrene, Poros—a copolymer ofpolystyrene/divinylbenzene, or reversibly cross-linked acrylamide. Manyother solid supports are commercially available and amenable to use inattaching oligonucleotides thereto.

In some embodiments, a solid support is coated with a material to aid inthe attachment of an oligonucleotide primer to the surface. Exemplarysurface coatings include, but are not limited to metals such as gold,silver, steel, aluminum, silicon, and copper.

In some embodiments, the solid support is a bead (e.g., silica gel,glass (e.g., controlled pore glass), magnetic bead, plastic, metal,polystyrene, or polymer bead). In some embodiments, the bead has a sizeof about 1 μm to about 100 μm in diameter. Bead diameters may beselected based on the sizes of the partitions (e.g., the sizes ofmicrofluidic channels or droplets as discussed herein).

Particles comprising oligonucleotides conjugated to a solid supportsurface, including barcode-labeled oligonucleotides, and methods ofmaking such particles, are known in the art. See, e.g., U.S. Pat. No.6,133,436; US 2011/0028334; and International Application No.PCT/US2015/037525, incorporated by reference herein.

Substrates Comprising Barcode Sequences

In some embodiments, substrates comprising a barcode sequence orrepeating clonal barcode sequences are provided. In some embodiments, asubstrate comprises a single barcode sequence (i.e., does not compriserepeating clonal barcode sequences). In some embodiments, the substratecomprising the single barcode sequence is a hairpin molecule. In someembodiments, the substrate comprising the single barcode sequence is alinear polynucleotide substrate. In some embodiments, the substratecomprising the single barcode sequence is a circular polynucleotidesubstrate. In some embodiments, the substrate comprising the singlebarcode sequence is encapsulated in a droplet.

In some embodiments, a substrate comprises repeating clonal barcodesequences. As used herein, a “substrate comprising repeating clonalbarcode sequences” refers to a composition that contains a plurality ofidentical “clonal” barcode sequences that are either physicallyconnected to each other (e.g., in a hairpin molecule, in a linearnucleic acid polymer, or in a circular nucleic acid polymer as tandemrepeating barcode sequences) or that are sequestered from othercomponents when delivered to a partition (e.g., encapsulated within adroplet that is delivered to a partition). In some embodiments, theclonal barcode sequences are not available to associate witholigonucleotide primers of particles in a partition without first beingreleased from the physical connection or sequestration. In someembodiments, a plurality of substrates (e.g., a plurality of hairpinmolecules, linear nucleic acid polymers, circular nucleic acid polymers,or droplets) are delivered to a partition. For example, in someembodiments, a partition comprises at least about 5, 10, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200 or more substrates.

In some embodiments, a substrate barcode sequence has a length of atleast 6 nucleotides. In some embodiments, the substrate barcode sequencehas a length of at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In some embodiments,the substrate barcode sequence has a length of about 6 to about 25nucleotides.

In some embodiments, the clonal barcode sequences from a particularsubstrate comprise a region of nucleotide sequence that is a uniqueidentifier sequence for all of the clonal barcode sequences from thatsubstrate, and the clonal barcode sequences further comprise apoly-thymine region and/or a poly-adenine region flanking the region ofunique identifier sequence. In some embodiments, a poly-adenine regionor poly-thymine comprises from about 15 to about 20, or from about 15 toabout 35 thymine nucleotides. In some embodiments, a poly-adenine regionof a clonal barcode sequence is used for hybridizing the clonal barcodesequence to an oligonucleotide primer at a poly-thymine region of theoligonucleotide primer. See, e.g., FIG. 1 .

In some embodiments, a barcode sequence or clonal barcode sequences froma particular substrate comprise a region of nucleotide sequence that isa unique identifier sequence for all of the clonal barcode sequencesfrom that substrate, and further comprise one or more universal tagsequences. In some embodiments, the universal tag sequence is used forhybridizing the substrate barcode sequence to an oligonucleotideconjugated to a particle.

In some embodiments, a substrate barcode sequence is a contiguitypreserved tagmented polynucleotide (e.g., DNA) sequence. In contiguitypreserved transposition or tagmentation, a tagmentase or transposase(e.g., Tn5 transposase) is used to modify DNA with adaptor sequenceswhile maintaining contiguity of DNA segments. The DNA can also belabeled or modified with barcode or index sequences. Methods ofpreparing contiguity preserved tagmented polynucleotide sequences areknown. See, e.g., Amini et al., Nature Genetics, 2014, 46:1343-1349; WO2016/061517; and U.S. Provisional Patent Application No. 62/436,288;each of which is incorporated by reference herein.

In some embodiments, a substrate comprising repeating clonal barcodesequences is a hairpin molecule of tandem repeating clonal barcodesequences, wherein the clonal barcode sequences are identical to eachother and wherein the barcode sequences are separated by a cleavablelinker between each repeating sequence. In some embodiments, the hairpinmolecule comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100 or more tandem repeating clonal barcodesequences.

In some embodiments, a substrate comprising repeating clonal barcodesequences is a linear nucleic acid polymer of tandem repeating clonalbarcode sequences, wherein the clonal barcode sequences are identical toeach other and wherein the barcode sequences are separated by acleavable linker between each repeating sequence. In some embodiments,the linear polymer comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more tandem repeatingclonal barcode sequences. In some embodiments, the linear nucleic acidpolymer comprises DNA, RNA, or a hybrid of DNA and RNA. In someembodiments, the linear nucleic acid polymer is double stranded. In someembodiments, the linear nucleic acid polymer is single stranded.

In some embodiments, a substrate comprising repeating clonal barcodesequences is a circular nucleic acid polymer of tandem repeating clonalbarcode sequences, wherein the clonal barcode sequences are identical toeach other and wherein the barcode sequences are separated by acleavable linker between each repeating sequence. In some embodiments,the circular polymer comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more tandem repeatingclonal barcode sequences. In some embodiments, the circular polymer is aplasmid. In some embodiments, the circular polymer is a DNA nanoball(i.e., a single stranded DNA molecule that collapses into a spheroidstructure due to secondary structures forming at regular intervals). Insome embodiments, the circular polymer is a multiple displacementamplified branched substrate. In some embodiments, the circular nucleicacid polymer comprises DNA, RNA, or a hybrid of DNA and RNA. In someembodiments, the circular nucleic acid polymer is double stranded. Insome embodiments, the circular nucleic acid polymer is single stranded.

In some embodiments, the substrate comprising the barcode sequence orrepeating clonal barcode sequences is a droplet encapsulating therepeating clonal barcode sequences. In some embodiments, the dropletencapsulating the repeating clonal barcode sequences comprises at least2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100 or more clonal barcode sequences that are identical to each other.In some embodiments, the droplet comprises an emulsion composition,i.e., a mixture of immiscible fluids (e.g., water and oil). In someembodiments, the droplet is an aqueous droplet that is surrounded by animmiscible carrier fluid (e.g., oil). In some embodiments, the dropletis an oil droplet that is surrounded by an immiscible carrier fluid(e.g., an aqueous solution). In some embodiments, the droplet has adiameter of about 0.001 microns to about 500 microns, e.g., about 0.005to about 100 microns, or about 0.01 to about 50 microns. The size of thedroplet may be selected based on the sizes of the partitions (e.g., thesizes of microfluidic channels or droplets as discussed herein). Methodsof generating droplets are described below and are also described, e.g.,in published patent applications WO 2011/109546 and WO 2012/061444, theentire content of each of which is incorporated by reference herein.

In some embodiments, wherein the substrate comprising a barcode sequenceor repeating clonal barcode sequences is a droplet encapsulating therepeating clonal barcode sequences, the droplet that is within apartition is relatively unstable and can be triggered to release thebarcode sequences into the partition without breaking the partition. Insome embodiments, factors such as surfactants, oils, osmolarity, or heatlability can affect the ability of a droplet within a partition torelease the contents of the droplet into the partition.

Partitioning

In some embodiments, a sample (e.g., a sample comprising target nucleicacids) is partitioned into a plurality of partitions. In someembodiments, the sample comprising target nucleic acids is partitionedsuch that the partition contains 0, 1, or more than 1 target nucleicacids. In some embodiments, the sample comprising target nucleic acidsis partitioned such that, on average, the partitions contain no morethan 1 target nucleic acid. In some embodiments, a sample forpartitioning further comprises particles conjugated to oligonucleotideprimers as described herein. In some embodiments, wherein the samplecomprises particles conjugated to oligonucleotide primers, the sample ispartitioned such that, on average, a partition contains about oneparticle. In some embodiments, wherein the sample comprises particlesconjugated to oligonucleotide primers, the sample is partitioned suchthat, on average, a partition contains at least two particles (e.g.,such that, on average, a partition contains two particles or containsthree particles). In some embodiments, a sample for partitioning furthercomprises substrates comprising a barcode sequence or repeating clonalbarcode sequences as described herein. In some embodiments, a sample forpartitioning further comprises one or more additional components,including but not limited to reagents for extension, ligation, reversetranscription, or amplification reactions (e.g., polymerases,nucleotides, buffers, salts, etc.).

Partitions can include any of a number of types of partitions, includingsolid partitions (e.g., wells or tubes) and fluid partitions (e.g.,aqueous droplets within an oil phase). In some embodiments, thepartitions are droplets. In some embodiments, the partitions aremicrochannels. Methods and compositions for partitioning a sample aredescribed, for example, in published patent applications WO 2010/036352,US 2010/0173394, US 2011/0092373, WO 2011/120024, and US 2011/0092376,the entire content of each of which is incorporated by reference herein.

In some embodiments, a sample (e.g., a sample comprising one or moretarget nucleic acids, particles conjugated to oligonucleotide primers,and/or substrates comprising repeating clonal barcode sequences) ispartitioned into a plurality of droplets. In some embodiments, a dropletcomprises an emulsion composition, i.e., a mixture of immiscible fluids(e.g., water and oil). In some embodiments, a droplet is an aqueousdroplet that is surrounded by an immiscible carrier fluid (e.g., oil).In some embodiments, a droplet is an oil droplet that is surrounded byan immiscible carrier fluid (e.g., an aqueous solution). In someembodiments, the droplets are relatively stable and have minimalcoalescence between two or more droplets. In some embodiments, less than0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, or 10% of droplets generated from a samplecoalesce with other droplets. The emulsions can also have limitedflocculation, a process by which the dispersed phase comes out ofsuspension in flakes. Methods of emulsion formation are described, forexample, in published patent applications WO 2011/109546 and WO2012/061444, the entire content of each of which is incorporated byreference herein.

In some embodiments, the droplet is formed by flowing an oil phasethrough an aqueous sample comprising the polynucleotide fragments andddPCR reaction components. The oil phase may comprise a fluorinated baseoil which may additionally be stabilized by combination with afluorinated surfactant such as a perfluorinated polyether. In someembodiments, the base oil comprises one or more of a HFE 7500, FC-40,FC-43, FC-70, or another common fluorinated oil. In some embodiments,the oil phase comprises an anionic fluorosurfactant. In someembodiments, the anionic fluorosurfactant is Ammonium Krytox(Krytox-AS), the ammonium salt of Krytox FSH, or a morpholino derivativeof Krytox FSH. Krytox-AS may be present at a concentration of about0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%,or 4.0% (w/w). In some embodiments, the concentration of Krytox-AS isabout 1.8%. In some embodiments, the concentration of Krytox-AS is about1.62%. Morpholino derivative of Krytox FSH may be present at aconcentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%,0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w/w). In some embodiments, theconcentration of morpholino derivative of Krytox FSH is about 1.8%. Insome embodiments, the concentration of morpholino derivative of KrytoxFSH is about 1.62%.

In some embodiments, the oil phase further comprises an additive fortuning the oil properties, such as vapor pressure, viscosity, or surfacetension. Non-limiting examples include perfluorooctanol and1H,1H,2H,2H-Perfluorodecanol. In some embodiments,1H,1H,2H,2H-Perfluorodecanol is added to a concentration of about 0.05%,0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1.0%, 1.25%, 1.50%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, or 3.0%(w/w). In some embodiments, 1H,1H,2H,2H-Perfluorodecanol is added to aconcentration of about 0.18% (w/w).

In some embodiments, the emulsion is formulated to produce highlymonodisperse droplets having a liquid-like interfacial film that can beconverted by heating into microcapsules having a solid-like interfacialfilm; such microcapsules may behave as bioreactors able to retain theircontents through an incubation period. The conversion to microcapsuleform may occur upon heating. For example, such conversion may occur at atemperature of greater than about 40°, 50°, 60°, 70°, 80°, 90°, or 95°C. During the heating process, a fluid or mineral oil overlay may beused to prevent evaporation. Excess continuous phase oil may or may notbe removed prior to heating. The biocompatible capsules may be resistantto coalescence and/or flocculation across a wide range of thermal andmechanical processing. Following conversion, the microcapsules may bestored at about −70°, −20°, 0°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°,20°, 25°, 30°, 35°, or 40° C.

The microcapsule partitions, which may contain one or morepolynucleotide sequences and/or one or more one or more sets of primerspairs, may resist coalescence, particularly at high temperatures.Accordingly, the capsules can be incubated at a very high density (e.g.,number of partitions per unit volume). In some embodiments, greater than100,000, 500,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 5,000,000,or 10,000,000 partitions may be incubated per mL. In some embodiments,the sample-probe incubations occur in a single well, e.g., a well of amicrotiter plate, without inter-mixing between partitions. Themicrocapsules may also contain other components necessary for theincubation.

In some embodiments, a sample (e.g., a sample comprising one or moretarget nucleic acids, particles conjugated to oligonucleotide primers,and/or substrates comprising a barcode sequence or repeating clonalbarcode sequences) is partitioned into at least 500 partitions, at least1000 partitions, at least 2000 partitions, at least 3000 partitions, atleast 4000 partitions, at least 5000 partitions, at least 6000partitions, at least 7000 partitions, at least 8000 partitions, at least10,000 partitions, at least 15,000 partitions, at least 20,000partitions, at least 30,000 partitions, at least 40,000 partitions, atleast 50,000 partitions, at least 60,000 partitions, at least 70,000partitions, at least 80,000 partitions, at least 90,000 partitions, atleast 100,000 partitions, at least 200,000 partitions, at least 300,000partitions, at least 400,000 partitions, at least 500,000 partitions, atleast 600,000 partitions, at least 700,000 partitions, at least 800,000partitions, at least 900,000 partitions, at least 1,000,000 partitions,at least 2,000,000 partitions, at least 3,000,000 partitions, at least4,000,000 partitions, at least 5,000,000 partitions, at least 10,000,000partitions, at least 20,000,000 partitions, at least 30,000,000partitions, at least 40,000,000 partitions, at least 50,000,000partitions, at least 60,000,000 partitions, at least 70,000,000partitions, at least 80,000,000 partitions, at least 90,000,000partitions, at least 100,000,000 partitions, at least 150,000,000partitions, or at least 200,000,000 partitions.

In some embodiments, a sample (e.g., a sample comprising one or moretarget nucleic acids, particles conjugated to oligonucleotide primers,and/or substrates comprising a barcode sequence or repeating clonalbarcode sequences) is partitioned into a sufficient number of partitionssuch that at least a majority of partitions have at least about 0.1 butno more than about 10 target nucleic acids per partition (e.g., about0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 targets perpartition). In some embodiments, at least a majority of the partitionshave at least about 0.1 but no more than about 5 targets per partition(e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, or 5 targets perpartition). In some embodiments, at least a majority of partitions haveno more than about 1 target nucleic acid per partition. In someembodiments, on average about 0.1, 0.2, 0.3, 0.4, 0.5, or 1 targetnucleic acids are present in each partition.

In some embodiments, the droplets that are generated are substantiallyuniform in shape and/or size. For example, in some embodiments, thedroplets are substantially uniform in average diameter. In someembodiments, the droplets that are generated have an average diameter ofabout 0.001 microns, about 0.005 microns, about 0.01 microns, about 0.05microns, about 0.1 microns, about 0.5 microns, about 1 microns, about 5microns, about 10 microns, about 20 microns, about 30 microns, about 40microns, about 50 microns, about 60 microns, about 70 microns, about 80microns, about 90 microns, about 100 microns, about 150 microns, about200 microns, about 300 microns, about 400 microns, about 500 microns,about 600 microns, about 700 microns, about 800 microns, about 900microns, or about 1000 microns. In some embodiments, the droplets thatare generated have an average diameter of less than about 1000 microns,less than about 900 microns, less than about 800 microns, less thanabout 700 microns, less than about 600 microns, less than about 500microns, less than about 400 microns, less than about 300 microns, lessthan about 200 microns, less than about 100 microns, less than about 50microns, or less than about 25 microns. In some embodiments, thedroplets that are generated are non-uniform in shape and/or size.

In some embodiments, the droplets that are generated are substantiallyuniform in volume. For example, in some embodiments, the droplets thatare generated have an average volume of about 0.001 nL, about 0.005 nL,about 0.01 nL, about 0.02 nL, about 0.03 nL, about 0.04 nL, about 0.05nL, about 0.06 nL, about 0.07 nL, about 0.08 nL, about 0.09 nL, about0.1 nL, about 0.2 nL, about 0.3 nL, about 0.4 nL, about 0.5 nL, about0.6 nL, about 0.7 nL, about 0.8 nL, about 0.9 nL, about 1 nL, about 1.5nL, about 2 nL, about 2.5 nL, about 3 nL, about 3.5 nL, about 4 nL,about 4.5 nL, about 5 nL, about 5.5 nL, about 6 nL, about 6.5 nL, about7 nL, about 7.5 nL, about 8 nL, about 8.5 nL, about 9 nL, about 9.5 nL,about 10 nL, about 11 nL, about 12 nL, about 13 nL, about 14 nL, about15 nL, about 16 nL, about 17 nL, about 18 nL, about 19 nL, about 20 nL,about 25 nL, about 30 nL, about 35 nL, about 40 nL, about 45 nL, orabout 50 nL. In some embodiments, the droplets have an average volume ofabout 50 picoliters to about 2 nanoliters. In some embodiments, thedroplets have an average volume of about 0.5 nanoliters to about 50nanoliters. In some embodiments, the droplets have an average volume ofabout 0.5 nanoliters to about 2 nanoliters.

Release of Barcode Sequences from Partitions

In some embodiments, after the step of partitioning the sample,particles conjugated to oligonucleotide primers, substrates comprising abarcode sequence or repeating clonal barcode sequences, and/or any othercomponents (e.g., reagents for amplification or polymerizationreactions), the individual barcode sequences are released from thesubstrate comprising the barcode sequence or repeating clonal barcodesequence into the partition. In some embodiments, the step of releasingthe barcode sequence or repeating clonal barcode sequences comprisestriggering the substrate (e.g., a droplet) to release the plurality ofclonal barcode sequences.

In some embodiments, barcode sequences are released from a droplet bybreaking the droplet. In some embodiments, heat is used to break thedroplet. In some embodiments, a photochemical reaction is used to breakthe droplet. In some embodiments, acoustic waves are used to break thedroplet. In some embodiments, a chemical reaction upon mixing thedroplet with the larger partition (e.g., a larger droplet) results inthe breaking of the smaller droplet.

In some embodiments, the substrate comprises repeating clonal barcodesequences, and the step of releasing the barcode sequences from thesubstrate comprises separating the repeating clonal barcode sequenceinto a plurality of clonal barcode sequences. In some embodiments, theindividual clonal barcode sequences are released from a hairpinmolecule, linear nucleic acid polymer substrate, or circular nucleicacid polymer substrate by cleaving the hairpin molecule, linear nucleicacid polymer substrate, or circular nucleic acid polymer at thecleavable linker or linkers between the repeating clonal barcodesequences. In some embodiments, the cleavable linker is a restrictionenzyme site that is cleaved by a restriction enzyme (e.g., anendonuclease such as a Type II endonuclease or Type IIS endonuclease).For example, in some embodiments, the cleavable linker comprises a TypeII restriction enzyme binding site (e.g., HhaI, HindIII, NotI, BbvCI,EcoRI, BglI) or a Type IIS restriction enzyme binding site (e.g., FokI,AlwI, BspMI, MnII, BbvI, BccI, MboI). In some embodiments, the cleavablelinker comprises a uridine incorporated site in a portion of anucleotide sequence. A uridine incorporated site can be cleaved, forexample, using a uracil glycosylase enzyme (e.g., a uracil N-glycosylaseenzyme or uracil DNA glycosylase enzyme). In some embodiments, thecleavable linker comprises a photocleavable nucleotide. Photocleavablenucleotides include, for example, photocleavable fluorescent nucleotidesand photocleavable biotinylated nucleotides. See, e.g., Li et al., PNAS,2003, 100:414-419; Luo et al., Methods Enzymol, 2014, 549:115-131.

Associating Substrate Barcode Sequences with Oligonucleotide Primers onParticles

After the substrate barcode sequences are released into the partition,the substrate barcode sequences are associated with particles in thepartition in order to generate a nucleic acid signature for theparticles in the partition. In some embodiments, a substrate barcodesequence can be associated with two particles located in the samepartition, resulting in the virtual joining of the two particles via thesubstrate barcode sequence. Accordingly, in some embodiments, the methodcomprises associating a substrate barcode sequence with a first particleconjugated to oligonucleotide primers comprising a first barcodesequence and a second particle conjugated to oligonucleotide primerscomprising a second barcode sequence. In some embodiments, wherein thepartition comprises three particles, a first substrate barcode sequencecan be associated with a first particle conjugated to oligonucleotideprimers comprising a first barcode sequence and a second particleconjugated to oligonucleotide primers comprising a second barcodesequence, and a second substrate barcode sequence can be associated witha third particle conjugated to oligonucleotide primers comprising athird barcode sequence.

In some embodiments, the method comprises associating two or moredistinct substrate barcode sequences (i.e., a first substrate barcodesequence from a first substrate and a second substrate barcode sequencefrom a second substrate) with two or more distinct particles conjugatedto oligonucleotide primers (e.g., a first particle conjugated tooligonucleotide primers comprising a first barcode sequence, a secondparticle conjugated to oligonucleotide primers comprising a secondbarcode sequence, and/or a third particle conjugated to oligonucleotideprimers comprising a third barcode sequence). Accordingly, in someembodiments, the associating step comprises associating (1) the firstparticle conjugated to oligonucleotide primers comprising the firstbarcode sequence with a first substrate barcode sequence, and (2) thesecond particle conjugated to oligonucleotide primers comprising thesecond barcode sequence with a second substrate barcode sequence. Insome embodiments, the associating step comprises associating (1) thefirst particle conjugated to oligonucleotide primers comprising thefirst barcode sequence with a first substrate barcode sequence; (2) thesecond particle conjugated to oligonucleotide primers comprising thesecond barcode sequence with a second substrate barcode sequence; and(3) the third particle conjugated to oligonucleotide primers comprisingthe third barcode sequence with a third substrate barcode sequence.

In some embodiments, the associating step comprises annealing asubstrate barcode sequence (e.g., a clonal barcode sequence) to anoligonucleotide primer conjugated to a particle and extending theannealed products with a polymerase. In some embodiments, the annealingcomprises hybridizing a poly-adenine region on a clonal barcode sequenceto a poly-thymine region on an oligonucleotide primer conjugated to theparticle. See, e.g., FIG. 1 . In some embodiments, at least 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 cycles of annealing and extension are performed toattach the substrate barcode sequences to the oligonucleotide primers.In some embodiments, at least 1 cycle of annealing and extension isperformed. In some embodiments, up to 5 cycles or up to 10 cycles ofannealing and extension are performed. Annealing and extension methodsare described in the art. See, e.g., US 2015/0284712 and US2015/0322503.

In some embodiments, the associating step comprises hybridizing a clonalbarcode sequence to an oligonucleotide primer conjugated to a particlein a partition, then pooling a plurality of partitions and extending thehybridized products with a polymerase in a bulk reaction. Hybridizationmethods are described in the art. See, e.g., International ApplicationNo. PCT/US2015/037525, incorporated by reference herein.

In some embodiments, wherein the substrate comprises a single barcodesequence, the associating step comprises (1) annealing anoligonucleotide primer of the first particle to the substrate barcodesequence and extending the annealed product with a polymerase, and (2)annealing an oligonucleotide primer of the second particle to thesubstrate barcode sequence and extending the annealed product with thepolymerase.

In some embodiments, the extension and amplification reaction comprisesthe use of a polymerase, e.g., a DNA polymerase. DNA polymerases for usein the methods described herein can be any polymerase capable ofreplicating a DNA molecule. In some embodiments, the DNA polymerase is athermostable polymerase. Thermostable polymerases are isolated from awide variety of thermophilic bacteria, such as Thermus aquaticus (Taq),Pyrococcus furiosus (Pfu), Pyrococcus woesei (Pwo), Bacillussterothermophilus (Bst), Sulfolobus acidocaldarius (Sac) Sulfolobussolfataricus (Sso), Pyrodictium occultum (Poc), Pyrodictium abyssi(Pab), and Methanobacterium thermoautotrophicum (Mth), as well as otherspecies. DNA polymerases are known in the art and are commerciallyavailable. In some embodiments, the DNA polymerase is Taq, Tbr, Tfl,Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso,Poc, Pab, Mth, Pho, ES4, VENT™, DEEPVENT™, or an active mutant, variant,or derivative thereof. In some embodiments, the polymerase is Taq DNApolymerase. In some embodiments, the polymerase is a high fidelity DNApolymerase (e.g., iProofrM High-Fidelity DNA Polymerase, Phusion®High-Fidelity DNA polymerase, Q5® High-Fidelity DNA polymerase,Platinum® Taq High Fidelity DNA polymerase, Accura® High-FidelityPolymerase). In some embodiments, the polymerase is a fast-startpolymerase (e.g., FastStart™ Taq DNA polymerase or FastStart™ HighFidelity DNA polymerase). In some embodiments, the polymerase is astrand displacing polymerase (e.g., phi29, or Bst DNA Polymerase, LargeFragment).

In some embodiments, the associating step comprises ligating a substratebarcode sequence (e.g., a clonal barcode sequence) to an oligonucleotideprimer of a first particle and to an oligonucleotide primer of a secondparticle. In some embodiments, the clonal barcode sequence and/oroligonucleotide primer sequence to be annealed are single-strandednucleic acid. In some embodiments, the ligation reaction comprises theuse of a ligase, e.g., a DNA ligase. Exemplary ligases for use in themethods described herein include, but are not limited to, T4 DNA ligaseand T4 RNA ligase. Nucleic acid ligation methods are described in theart; see, e.g., Li et al., Anal Biochem, 2006, 349:242-246; and Kuhn etal., FEBS J., 2005, 212:5991-6000.

Downstream Applications

Once the particles conjugated to oligonucleotide primers are associatedwith substrate barcode sequences in order to generate a unique nucleicacid signature for the particles within a specific partition, thenucleic acid signature can be used for deconvoluting data generated indownstream applications, such as downstream detection and/or analysismethods. In some embodiments, the downstream application is sequencing(e.g., high throughput sequencing).

In some embodiments, after the step of associating the substrate barcodesequences with particles in the partition in order to generate a nucleicacid signature for the particles in the partition, the method furthercomprises associating a target nucleic acid in the partition with aparticle in the partition. In some embodiments, the step of associatinga target nucleic acid in the partition with a particle in the partitioncomprises hybridizing a target nucleic acid, or a portion thereof, to aportion of an oligonucleotide primer conjugated to a particle. In someembodiments, the target nucleic acid or portion thereof hybridizes to auniversal tag portion or to a random sequence portion of theoligonucleotide primer.

In some embodiments, the step of associating a target nucleic acid in apartition with a particle in the partition is carried out prior to thestep of associating the substrate barcode sequences with particles inthe partition. In some embodiments, while in partition, a target nucleicacid or a portion thereof is hybridized to a portion of anoligonucleotide primer conjugated to a particle, and the clonal barcodesequence is also hybridized to a portion of the oligonucleotide primerconjugated to the particle. The partitions are then broken and thecontents of multiple partitions are pooled before performing anextension reaction to extend the hybridized target nucleicacid-oligonucleotide primer product and the hybridized substrate barcodesequence-oligonucleotide primer product.

In some embodiments, the method further comprises polymerizing thehybridized target nucleic acid-oligonucleotide primer product. In someembodiments, the polymerization comprises primer extension. In someembodiments, the polymerization comprises reverse transcription (e.g.,reverse transcription of a RNA target nucleic acid). In someembodiments, the method further comprises amplifying the target nucleicacid-oligonucleotide primer product. In some embodiments, theamplification reaction is a droplet digital PCR reaction. Methods forperforming PCR in droplets are described, for example, in US2014/0162266, US 2014/0302503, and US 2015/0031034, the contents of eachof which is incorporated by reference.

Release of Partition Contents

In some embodiments, after the particles conjugated to oligonucleotideprimers are associated with substrate barcode sequences in order togenerate a unique nucleic acid signature for each partition, thecontents of the partitions (e.g., target nucleic acids associated with aparticle as described herein) are released prior to the downstreamapplication, e.g., to pool multiple partitions for a downstreamapplication such as a sequencing reaction. Partition breaking can beaccomplished by any of a number of methods, including but not limited toelectrical methods and introduction of a destabilizing fluid. See, e.g.,Zeng et al., Anal Chem 2011, 83:2083-2089. Methods of breakingpartitions are also described, for example, in US 2013/0189700,incorporated by reference herein.

In some embodiments, partitions are broken by mixing the partitions(e.g., droplets) with a destabilizing fluid. In some embodiments, thedestabilizing fluid is chloroform. In some embodiments, thedestabilizing fluid comprises a perfluorinated alcohol. In someembodiments, the destabilizing fluid comprises a fluorinated oil, suchas a perfluorocarbon oil.

In some embodiments, the method further comprises purifying a targetnucleic acid that is released from a partition (e.g., a target nucleicacid associated with a particle as described herein), e.g., in order toseparate the target nucleic acid from other partition components. Insome embodiments, the purifying step comprises the use of solid-phasereversible immobilization (SPRI) paramagnetic bead reagents. SPRIparamagnetic bead reagents are commercially available, for example inthe Agencourt AMPure XP PCR purification system (Beckman-Coulter, Brea,Calif.).

Sequencing

In some embodiments, a target nucleic acid from a partition having aunique nucleic acid signature as described herein is analyzed by asequencing or genotyping method. In some embodiments, the target nucleicacid is analyzed by sequencing, e.g., high throughput sequencing. Insome embodiments, the method of analyzing a partitioned sample (e.g., acell or target nucleic acid) further comprises determining the nucleicacid signatures of sequence reads and deconvoluting the nucleic acidsignatures in order to allow sequence information from each partitionedsample to be uniquely identified.

Methods for high throughput sequencing and genotyping are known in theart. For example, such sequencing technologies include, but are notlimited to, pyrosequencing, sequencing-by-ligation, single moleculesequencing, sequence-by-synthesis (SBS), massive parallel clonal,massive parallel single molecule SBS, massive parallel single moleculereal-time, massive parallel single molecule real-time nanoporetechnology, etc. Morozova and Marra provide a review of some suchtechnologies in Genomics, 92: 255 (2008), herein incorporated byreference in its entirety.

Exemplary DNA sequencing techniques include fluorescence-basedsequencing methodologies (See, e.g., Birren et al., Genome Analysis:Analyzing DNA, 1, Cold Spring Harbor, N.Y.; herein incorporated byreference in its entirety). In some embodiments, automated sequencingtechniques understood in that art are utilized. In some embodiments, thepresent technology provides parallel sequencing of partitioned amplicons(PCT Publication No. WO 2006/0841,32, herein incorporated by referencein its entirety). In some embodiments, DNA sequencing is achieved byparallel oligonucleotide extension (See, e.g., U.S. Pat. Nos. 5,750,341;and 6,306,597, both of which are herein incorporated by reference intheir entireties). Additional examples of sequencing techniques includethe Church polony technology (Mitra et al., 2003, AnalyticalBiochemistry 320, 55-65; Shendure et al., 2005 Science 309, 1728-1732;and U.S. Pat. Nos. 6,432,360; 6,485,944; 6,511,803; herein incorporatedby reference in their entireties), the 454 picotiter pyrosequencingtechnology (Margulies et al., 2005 Nature 437, 376-380; U.S. PublicationNo. 2005/0130173; herein incorporated by reference in their entireties),the Solexa single base addition technology (Bennett et al., 2005,Pharmacogenomics, 6, 373-382; U.S. Pat. Nos. 6,787,308; and 6,833,246;herein incorporated by reference in their entireties), the Lynxmassively parallel signature sequencing technology (Brenner et al.(2000). Nat. Biotechnol. 18:630-634; U.S. Pat. Nos. 5,695,934;5,714,330; herein incorporated by reference in their entireties), andthe Adessi PCR colony technology (Adessi et al. (2000). Nucleic AcidRes. 28, E87; WO 2000/018957; herein incorporated by reference in itsentirety).

Typically, high throughput sequencing methods share the common featureof massively parallel, high-throughput strategies, with the goal oflower costs in comparison to older sequencing methods (See, e.g.,Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al.,Nature Rev. Microbiol., 7:287-296; each herein incorporated by referencein their entirety). Such methods can be broadly divided into those thattypically use template amplification and those that do not.Amplification-requiring methods include pyrosequencing commercialized byRoche as the 454 technology platforms (e.g., GS 20 and GS FLX), theSolexa platform commercialized by Illumina, and the SupportedOligonucleotide Ligation and Detection (SOLiD) platform commercializedby Applied Biosystems. Non-amplification approaches, also known assingle-molecule sequencing, are exemplified by the HeliScope platformcommercialized by Helicos BioSciences, and platforms commercialized byVisiGen, Oxford Nanopore Technologies Ltd., Life Technologies/IonTorrent, and Pacific Biosciences, respectively.

In pyrosequencing (Voelkerding et al., Clinical Chem., 55: 641-658,2009; MacLean et al., Nature Rev. Microbial., 7:287-296; U.S. Pat. Nos.6,210,891; and 6,258,568; each herein incorporated by reference in itsentirety), template DNA is fragmented, end-repaired, ligated toadaptors, and clonally amplified in-situ by capturing single templatemolecules with beads bearing oligonucleotides complementary to theadaptors. Each bead bearing a single template type is compartmentalizedinto a water-in-oil microvesicle, and the template is clonally amplifiedusing a technique referred to as emulsion PCR. The emulsion is disruptedafter amplification and beads are deposited into individual wells of apicotitre plate functioning as a flow cell during the sequencingreactions. Ordered, iterative introduction of each of the four dNTPreagents occurs in the flow cell in the presence of sequencing enzymesand luminescent reporter such as luciferase. In the event that anappropriate dNTP is added to the 3′ end of the sequencing primer, theresulting production of ATP causes a burst of luminescence within thewell, which is recorded using a CCD camera. It is possible to achieveread lengths greater than or equal to 400 bases, and 10⁶ sequence readscan be achieved, resulting in up to 500 million base pairs (Mb) ofsequence.

In the Solexa/Illumina platform (Voelkerding et al., Clinical Chem., 55.641-658, 2009; MacLean et al., Nature Rev. Microbial., 7:287-296; U.S.Pat. Nos. 6,833,246; 7,115,400; and 6,969,488; each herein incorporatedby reference in its entirety), sequencing data are produced in the formof shorter-length reads. In this method, single-stranded fragmented DNAis end-repaired to generate 5′-phosphorylated blunt ends, followed byKlenow-mediated addition of a single A base to the 3′ end of thefragments. A-addition facilitates addition of T-overhang adaptoroligonucleotides, which are subsequently used to capture thetemplate-adaptor molecules on the surface of a flow cell that is studdedwith oligonucleotide anchors. The anchor is used as a PCR primer, butbecause of the length of the template and its proximity to other nearbyanchor oligonucleotides, extension by PCR results in the “arching over”of the molecule to hybridize with an adjacent anchor oligonucleotide toform a bridge structure on the surface of the flow cell. These loops ofDNA are denatured and cleaved. Forward strands are then sequenced withreversible dye terminators. The sequence of incorporated nucleotides isdetermined by detection of post-incorporation fluorescence, with eachfluor and block removed prior to the next cycle of dNTP addition.Sequence read length ranges from 36 nucleotides to over 50 nucleotides,with overall output exceeding 1 billion nucleotide pairs per analyticalrun.

Sequencing nucleic acid molecules using SOLiD technology (Voelkerding etal., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.Microbial., 7:287-296; U.S. Pat. Nos. 5,912,148; and 6,130,073; eachherein incorporated by reference in their entirety) also involvesfragmentation of the template, ligation to oligonucleotide adaptors,attachment to beads, and clonal amplification by emulsion PCR. Followingthis, beads bearing template are immobilized on a derivatized surface ofa glass flow-cell, and a primer complementary to the adaptoroligonucleotide is annealed. However, rather than utilizing this primerfor 3′ extension, it is instead used to provide a 5′ phosphate group forligation to interrogation probes containing two probe-specific basesfollowed by 6 degenerate bases and one of four fluorescent labels. Inthe SOLiD system, interrogation probes have 16 possible combinations ofthe two bases at the 3′ end of each probe, and one of four fluors at the5′ end. Fluor color, and thus identity of each probe, corresponds tospecified color-space coding schemes. Multiple rounds (usually 7) ofprobe annealing, ligation, and fluor detection are followed bydenaturation, and then a second round of sequencing using a primer thatis offset by one base relative to the initial primer. In this manner,the template sequence can be computationally re-constructed, andtemplate bases are interrogated twice, resulting in increased accuracy.Sequence read length averages 35 nucleotides, and overall output exceeds4 billion bases per sequencing run.

In some embodiments, nanopore sequencing is employed (See, e.g., Astieret al., J. Am. Chem. Soc. 2006 Feb. 8; 128 (5) 1705-10, incorporated byreference). The theory behind nanopore sequencing relates to what occurswhen a nanopore is immersed in a conducting fluid and a potential(voltage) is applied across it. Under these conditions a slight electriccurrent due to conduction of ions through the nanopore can be observed,and the amount of current is exceedingly sensitive to the size of thenanopore. As each base of a nucleic acid passes through the nanopore,this causes a change in the magnitude of the current through thenanopore that is distinct for each of the four bases, thereby allowingthe sequence of the DNA molecule to be determined.

In some embodiments, HeliScope by Helicos BioSciences is employed(Voelkerding et al., Clinical Chem., 55. 641-658, 2009; MacLean et al.,Nature Rev. Microbial, 7:287-296; U.S. Pat. Nos. 7,169,560; 7,282,337;7,482,120; 7,501,245; 6,818,395; 6,911,345; and 7,501,245; each hereinincorporated by reference in their entirety). Template DNA is fragmentedand polyadenylated at the 3′ end, with the final adenosine bearing afluorescent label. Denatured polyadenylated template fragments areligated to poly(dT) oligonucleotides on the surface of a flow cell.Initial physical locations of captured template molecules are recordedby a CCD camera, and then label is cleaved and washed away. Sequencingis achieved by addition of polymerase and serial addition offluorescently-labeled dNTP reagents. Incorporation events result influor signal corresponding to the dNTP, and signal is captured by a CCDcamera before each round of dNTP addition. Sequence read length rangesfrom 25-50 nucleotides, with overall output exceeding 1 billionnucleotide pairs per analytical run.

The Ion Torrent technology is a method of DNA sequencing based on thedetection of hydrogen ions that are released during the polymerizationof DNA (See, e.g., Science 327(5970): 1190 (2010); U.S. Pat. Appl. Pub.Nos. 2009/0026082; 2009/0127589; 2010/0301398; 2010/0197507;2010/0188073; and 2010/0137143, incorporated by reference in theirentireties for all purposes). A microwell contains a template DNA strandto be sequenced. Beneath the layer of microwells is a hypersensitiveISFET ion sensor. All layers are contained within a CMOS semiconductorchip, similar to that used in the electronics industry. When a dNTP isincorporated into the growing complementary strand a hydrogen ion isreleased, which triggers the hypersensitive ion sensor. If homopolymerrepeats are present in the template sequence, multiple dNTP moleculeswill be incorporated in a single cycle. This leads to a correspondingnumber of released hydrogens and a proportionally higher electronicsignal. This technology differs from other sequencing technologies inthat no modified nucleotides or optics are used. The per base accuracyof the Ion Torrent sequencer is ˜99.6% for 50 base reads, with ˜100 Mbgenerated per run. The read-length is 100 base pairs. The accuracy forhomopolymer repeats of 5 repeats in length is ˜98%. The benefits of ionsemiconductor sequencing are rapid sequencing speed and low upfront andoperating costs.

Another exemplary nucleic acid sequencing approach that may be adaptedfor use with the present invention was developed by Stratos Genomics,Inc. and involves the use of Xpandomers. This sequencing processtypically includes providing a daughter strand produced by atemplate-directed synthesis. The daughter strand generally includes aplurality of subunits coupled in a sequence corresponding to acontiguous nucleotide sequence of all or a portion of a target nucleicacid in which the individual subunits comprise a tether, at least oneprobe or nucleobase residue, and at least one selectively cleavablebond. The selectively cleavable bond(s) is/are cleaved to yield anXpandomer of a length longer than the plurality of the subunits of thedaughter strand. The Xpandomer typically includes the tethers andreporter elements for parsing genetic information in a sequencecorresponding to the contiguous nucleotide sequence of all or a portionof the target nucleic acid. Reporter elements of the Xpandomer are thendetected. Additional details relating to Xpandomer-based approaches aredescribed in, for example, U.S. Pat. Pub No. 2009/0035777, which isincorporated herein in its entirety.

Other single molecule sequencing methods include real-time sequencing bysynthesis using a VisiGen platform (Voelkerding et al., Clinical Chem.,55: 641-58, 2009; U.S. Pat. No. 7,329,492; and U.S. patent appl. Ser.Nos. 11/671,956; and 11/781,166; each herein incorporated by referencein their entirety) in which immobilized, primed DNA template issubjected to strand extension using a fluorescently-modified polymeraseand florescent acceptor molecules, resulting in detectible fluorescenceresonance energy transfer (FRET) upon nucleotide addition.

Another real-time single molecule sequencing system developed by PacificBiosciences (Voelkerding et al., Clinical Chem., 55. 641-658, 2009;MacLean et al., Nature Rev. Microbiol., 7:287-296; U.S. Pat. Nos.7,170,050; 7,302,146; 7,313,308; and 7,476,503; all of which are hereinincorporated by reference) utilizes reaction wells 50-100 nm in diameterand encompassing a reaction volume of approximately 20 zeptoliters(10⁻²¹ L). Sequencing reactions are performed using immobilizedtemplate, modified phi29 DNA polymerase, and high local concentrationsof fluorescently labeled dNTPs. High local concentrations and continuousreaction conditions allow incorporation events to be captured in realtime by fluor signal detection using laser excitation, an opticalwaveguide, and a CCD camera.

In some embodiments, the single molecule real time (SMRT) DNA sequencingmethods using zero-mode waveguides (ZMWs) developed by PacificBiosciences, or similar methods, are employed. With this technology, DNAsequencing is performed on SMRT chips, each containing thousands ofzero-mode waveguides (ZMWs). A ZMW is a hole, tens of nanometers indiameter, fabricated in a 100 nm metal film deposited on a silicondioxide substrate. Each ZMW becomes a nanophotonic visualization chamberproviding a detection volume of just 20 zeptoliters (10⁻²¹ L). At thisvolume, the activity of a single molecule can be detected amongst abackground of thousands of labeled nucleotides. The ZMW provides awindow for watching DNA polymerase as it performs sequencing bysynthesis. Within each chamber, a single DNA polymerase molecule isattached to the bottom surface such that it permanently resides withinthe detection volume. Phospholinked nucleotides, each type labeled witha different colored fluorophore, are then introduced into the reactionsolution at high concentrations which promote enzyme speed, accuracy,and processivity. Due to the small size of the ZMW, even at these highconcentrations, the detection volume is occupied by nucleotides only asmall fraction of the time. In addition, visits to the detection volumeare fast, lasting only a few microseconds, due to the very smalldistance that diffusion has to carry the nucleotides. The result is avery low background.

Processes and systems for such real time sequencing that may be adaptedfor use with the invention are described in, for example, U.S. Pat. Nos.7,405,281; 7,315,019; 7,313,308; 7,302,146; and 7,170,050; and U.S. Pat.Pub. Nos. 2008/0212960; 2008/0206764; 2008/0199932; 2008/0199874;2008/0176769; 2008/0176316; 2008/0176241; 2008/0165346; 2008/0160531;2008/0157005; 2008/0153100; 2008/0153095; 2008/0152281; 2008/0152280;2008/0145278; 2008/0128627; 2008/0108082; 2008/0095488; 2008/0080059;2008/0050747; 2008/0032301; 2008/0030628; 2008/0009007; 2007/0238679;2007/0231804; 2007/0206187; 2007/0196846; 2007/0188750; 2007/0161017;2007/0141598; 2007/0134128; 2007/0128133; 2007/0077564; 2007/0072196;and 2007/0036511; and Korlach et al. (2008), “Selective aluminumpassivation for targeted immobilization of single DNA polymerasemolecules in zero-mode waveguide nanostructures,” PNAS 105(4): 1176-81,all of which are herein incorporated by reference in their entireties.

III. Partition Libraries

In another aspect, partition libraries comprising a plurality ofpartitions (e.g., at least about 100; 200; 300; 500; 750; 1000; 2500;5000; 7500; 10,000; 15,000; 20,000; 30,000, or more partitions) areprovided. In some embodiments, at least some partitions comprise atleast a first particle conjugated to oligonucleotide primers comprisinga first barcode sequence and a second particle conjugated tooligonucleotide primers comprising a second barcode sequence. In someembodiments, the partitions comprise 0, 1, or more than 1 particles perpartition. In some embodiments, the partitions have an average of aboutone particle per partition. In some embodiments, the partitions have anaverage of about two particles per partition. In some embodiments, thepartitions have an average of about three particles per partition.

In some embodiments, at least some partitions of the partition library(e.g., a majority, substantially all, or all of the partitions of thepartition library) comprise at least a first particle conjugated tooligonucleotide primers comprising a first barcode sequence and a secondparticle conjugated to oligonucleotide primers comprising a secondbarcode sequence, and further comprise a substrate comprising a barcodesequence or repeating clonal barcode sequences. In some embodiments, atleast some partitions of the partition library comprise (1) twoparticles, wherein the first particle is conjugated to oligonucleotideprimers comprising a first barcode sequence and the second particleconjugated to oligonucleotide primers comprising a second barcodesequence; and (2) a substrate comprising a barcode sequence or repeatingclonal barcode sequences. In some embodiments, at least some partitionsof the partition library comprise (1) three particles, wherein the firstparticle is conjugated to oligonucleotide primers comprising a firstbarcode sequence, the second particle conjugated to oligonucleotideprimers comprising a second barcode sequence, and the third particle isconjugated to oligonucleotide primers comprising a third barcodesequence; and (2) at least two substrates, each comprising a barcodesequence or repeating clonal barcode sequences, wherein the barcodesequences of the first substrate and the second substrate aredistinguishable sequences. In some embodiments, a majority,substantially all, or all of the partitions of the partition librarycomprise at least one particles, at least two particles, or at leastthree particles. Particles having conjugated to oligonucleotide primersconjugated thereto and substrates comprising the barcode sequences orrepeating clonal barcode sequences are described in Section II above.

In some embodiments, at least some partitions of the partition librarycomprise at least a first particle conjugated to oligonucleotide primerscomprising a first barcode sequence and a second particle conjugated tooligonucleotide primers comprising a second barcode sequence, and atleast one substrate barcode sequence associated with the first particleand the second particle. In some embodiments, at least some partitionsof the partition library comprise (1) a first particle conjugated tooligonucleotide primers comprising a first barcode sequence with a firstsubstrate barcode sequence, and (2) a second particle conjugated tooligonucleotide primers comprising a second barcode sequence with asecond substrate barcode sequence. In some embodiments, a majority,substantially all, or all of the partitions of the partition librarycomprise at least two particles. In some embodiments, the firstsubstrate barcode sequence and the second substrate barcode sequence aredistinguishable sequences. In some embodiments, the first substratebarcode sequence and the second substrate barcode sequence are identicalsequences. In some embodiments, the substrate barcode sequences arecontiguity preserved tagmented polynucleotide (e.g., DNA) sequences.

In some embodiments, at least some partitions of the partition library(e.g., a majority, substantially all, or all of the partitions of thepartition library) comprise (1) a first particle conjugated tooligonucleotide primers comprising a first barcode sequence with a firstsubstrate barcode sequence; (2) a second particle conjugated tooligonucleotide primers comprising a second barcode sequence with asecond substrate barcode sequence; and (3) a third particle conjugatedto oligonucleotide primers comprising a third barcode sequence with athird substrate barcode sequence. In some embodiments, a majority,substantially all, or all of the partitions of the partition librarycomprise at least three particles.

In some embodiments, the substrate comprises repeating clonal barcodesequences. In some embodiments, the repeating clonal barcode sequencescomprise tandem repeating clonal barcode sequences that are separated bya cleavable linker (e.g., a hairpin molecule, a linear nucleic acidpolymer, or a circular nucleic acid polymer as described herein).

In some embodiments, the substrate comprising the barcode sequence orrepeating clonal barcode sequences is a droplet encapsulating thebarcode sequence or repeating clonal barcode sequences.

In some embodiments, at least some partitions of the partition library(e.g., a majority, substantially all, or all of the partitions of thepartition library) further comprise a sample (e.g., one or more targetnucleic acids, or one or more cells). In some embodiments, the samplecomprising target nucleic acids comprises DNA, RNA, or a combination orhybrid thereof. In some embodiments, the sample is a sample comprisingcells, e.g., is a single-cell sample. In some embodiments, the sample isa sample as described in Section II above.

In some embodiments, the partitions further comprise additional reagentsor components for polymerization, amplification, reverse transcription,or primer extension (e.g., polymerases, salts, nucleotides, buffers,stabilizers, primers, detectable agents, or nuclease-free water) asdescribed herein.

IV. Kits

In another aspect, kits for generating a nucleic acid signature foridentifying particles associated in a partition are provided. In someembodiments, a kit comprises:

-   -   (a) a plurality of particles comprising a solid support surface,        the solid support surface of a particle having a plurality of        oligonucleotide primers conjugated thereon, wherein the        oligonucleotide primers comprise a barcode sequence and wherein        at least a majority of the plurality of oligonucleotide primers        conjugated to a solid support surface comprise the same barcode        sequence; and    -   (b) a substrate comprising a barcode sequence or repeating        clonal barcode sequences.

Particles having conjugated to oligonucleotide primers conjugatedthereto and substrates comprising the barcode sequence or repeatingclonal barcode sequences are described in Section II above.

In some embodiments, the substrate comprises repeating clonal barcodesequences that comprise tandem repeating clonal barcode sequences thatare separated by a cleavable linker (e.g., a hairpin molecule, a linearnucleic acid polymer, or a circular nucleic acid polymer as describedherein). In some embodiments, the substrate comprising the barcodesequence or repeating clonal barcode sequences is a dropletencapsulating the repeating clonal barcode sequences.

In some embodiments, the kit further comprises one or more reagents forpolymerization, amplification, reverse transcription, or primerextension (e.g., polymerases, salts, nucleotides, buffers, stabilizers,primers, detectable agents, or nuclease-free water) as described herein.

In some embodiments, the kit further comprises instructions forperforming a method as described herein (e.g., instructions forpartitioning or instructions for associating substrate barcode sequenceswith particles conjugated to oligonucleotide primers).

VI. Examples

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1: Generation of Nucleic Acid Sequence for Virtually LinkingOligonucleotide-Loaded Beads in Partitions

In partition libraries that are not loaded with beads (such asoligonucleotide-loaded beads) in a deterministic fashion, beadconcentrations are typically adjusted so that only about 1 out of 10partitions are occupied by a bead, in order to ensure that partitionshave only 1 bead. This results in about 90% dead volume in thepartitions.

If bead concentration is adjusted resulting in either 2 or 3 beads perpartition on average, droplet occupancy increases to 85% and 95%,respectively. One benefit of such an approach is that dead volume isdrastically minimized. In such a high occupancy scheme, witholigonucleotide-loaded beads present at 1×-4× concentrations, verylittle detrimental effect is expected on molecular biology reactions(e.g., a reverse transcription reaction). However, it would bebeneficial to be able to deconvolute which beads are present together ina single partition (e.g., for deconvoluting sequencing data).

One approach for deconvoluting which beads are present together in asingle partition is to provide partitions with substrates comprisingbarcode sequences for generating a unique combination of sequences forbeads in a particular partition, such that upon their sequence analysis(e.g., by next-generation sequencing), the beads are virtually linked.An exemplary schematic of such an approach is shown in FIG. 1 . As shownin FIG. 1 , partitions (e.g., droplets) are loaded to have on average 2barcode-labeled oligonucleotide-loaded beads, resulting in 85% dropletoccupancy. Droplets are also loaded with substrates comprising repeatingclonal barcode sequences (as shown in FIG. 1A, hairpin substrates), forexample at a concentration of about 100-200 hairpin substrates perdroplet to ensure that each droplet is provided with sufficient hairpinsubstrates for attaching to the oligonucleotides of beads. For releasingthe clonal barcode sequences into separate sequences, the linkers in thehairpins are cleaved. The separated clonal barcode sequences aredenatured into single-stranded sequences, and then annealing andextension are carried out to attach the clonal barcode sequences to theoligonucleotides of the beads, thus creating unique combinations ofbarcode-labeled oligonucleotide-loaded beads and clonal barcodesequences. For example, as shown in FIG. 1C, by seeing that both beads 1and 2 are joined with clonal barcode sequence 5, one can deconvolutethat both beads occupied the same droplet.

As shown in FIG. 2 and FIG. 3 , a similar strategy of combiningoligonucleotides on beads and clonal barcode sequences can be carriedout using clonal barcode substrates that are circular substrates (FIG. 2) or linear substrates (FIG. 3 ), including single stranded nucleic acidpolymers (RNA, DNA, or a mixture thereof) and double stranded nucleicacid polymers (RNA, DNA, or a mixture thereof).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of generating a nucleic acid signaturefor identifying particles associated in a partition, the methodcomprising: (a) partitioning a sample into a plurality of partitions,wherein at least some of the partitions comprise (i) a cell comprisingtarget nucleic acids and (ii) a particle comprising a solid supportsurface, the solid support surface having a plurality of oligonucleotideprimers conjugated thereon, wherein the oligonucleotide primers comprisea barcode sequence that is unique to the particle and wherein at least amajority of the plurality of oligonucleotide primers conjugated to asolid support surface comprise the same barcode sequence; (b) providingin the partitions of step (a) one or more barcode sequences that arefrom a substrate comprising a barcode sequence or repeating clonalbarcode sequences, wherein the barcode sequence or repeating clonalbarcode sequences of the substrate comprise a region of nucleotidesequence that is a unique identifier sequence for the barcodesequence(s) from the substrate; and (c) for a partition from step (b)that comprises more than one particle, generating a nucleic acidsignature for the particles in the partition by attaching differentclonal copies of the barcode sequences from the substrate to (i) anoligonucleotide primer comprising a first barcode sequence that isconjugated to a first particle and (ii) an oligonucleotide primercomprising a second barcode sequence that is conjugated to a secondparticle; thereby generating a nucleic acid signature for the particlesthat are in the partition.
 2. The method of claim 1, wherein the targetnucleic acids are genomic DNA from the cell and the method compriseshybridizing the target nucleic acids, or a portion thereof, to a portionof a oligonucleotide primer from the particle thereby incorporating acopy of a barcode into the target nucleic acid or portion thereof. 3.The method of claim 2, wherein reverse transcription has occurred in thecells to form cDNA from RNA in the cell and the cDNA are the targetnucleic acids.
 4. The method of claim 3, wherein the reversetranscription occurs in the partitions using the oligonucleotide primersthereby incorporating a copy of a barcode into the cDNA.
 5. The methodof claim 1, wherein step (b) comprises releasing the barcode sequence orthe repeating clonal barcode sequences from the substrate.
 6. The methodof claim 5, wherein the substrate comprises tandem repeating clonalbarcode sequences that are separated by a cleavable linker, and whereinthe releasing comprises cleaving the substrate at the cleavable linker.7. The method of claim 5, wherein the substrate comprising the barcodesequence or the repeating clonal barcode sequences comprises a dropletencapsulating the barcode sequence or repeating clonal barcodesequences, and wherein the releasing comprises breaking the droplet. 8.The method of claim 1, wherein the substrate barcode sequence orrepeating clonal barcode sequences comprise DNA, RNA, or a DNA/RNAhybrid.
 9. The method of claim 1, wherein the substrate barcode sequenceis a contiguity preserved tagmented polynucleotide sequence.
 10. Themethod of claim 1, wherein the substrate comprises repeating clonalbarcode sequences.
 11. The method of claim 1, wherein the substratecomprises a single barcode sequence, and wherein step (c) comprises: (1)annealing an oligonucleotide primer of the first particle to thesubstrate barcode sequence and extending the annealed product with apolymerase, and (2) annealing an oligonucleotide primer of the secondparticle to the substrate barcode sequence and extending the annealedproduct with the polymerase.
 12. The method of claim 1, wherein step (c)comprises annealing the first substrate barcode sequence to anoligonucleotide primer of the first particle and the second substratebarcode sequence to an oligonucleotide primer of the second particle andextending the annealed products with a polymerase.
 13. The method ofclaim 1, wherein step (c) comprises ligating a substrate barcodesequence to both an oligonucleotide primer of the first particle and anoligonucleotide primer of the second particle.
 14. The method of claim1, wherein the partitions are droplets.